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|Ref Type||Journal Article|
|Authors||Ipsaro JJ, Shen C, Arai E, Xu Y, Kinney JB, Joshua-Tor L, Vakoc CR, Shi J|
|Title||Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis.|
|Abstract Text||Genetic alterations conferring resistance to the effects of chemical inhibitors are valuable tools for validating on-target effects in cells. Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel) mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors. We used this approach to identify novel resistance alleles of two lysine methyltransferases, DOT1L and EZH2, which are each essential for the growth of MLL-fusion leukemia cells. We biochemically characterized the DOT1L mutation, showing that it is significantly more active than the wild-type enzyme. These findings validate the on-target anti-leukemia activities of existing DOT1L and EZH2 inhibitors and reveal a simple method for deriving drug-resistance alleles for novel targets, which may have utility during early stages of drug development.|
|Molecular Profile||Treatment Approach|
|Gene Name||Source||Synonyms||Protein Domains||Gene Description||Gene Role|
|Therapy Name||Drugs||Efficacy Evidence||Clinical Trials|
|Drug Name||Trade Name||Synonyms||Drug Classes||Drug Description|
|Gene||Variant||Impact||Protein Effect||Variant Description||Associated with drug Resistance|
|DOT1L||V293_L296delinsMM||indel||gain of function||DOT1L V293_L296delinsMM results in a deletion of four amino acids in the DOT1 domain of the Dot1l protein from amino acids 293 to 296, combined with the insertion of two methionines (M) at the same site (UniProt.org). V293_L296delinsMM results in increased Dot1l lysine methyltransferase activity and decreased sensitivity to EPZ-5676 (pinometostat) in culture (PMID: 28231254).|
|EZH2||T678_R679delinsKK||indel||unknown||EZH2 T678_R679delinsKK results in a deletion of two amino acids in the SET domain of the Ezh2 protein from amino acids 678 to 679, combined with the insertion of two lysines (K) at the same site (UniProt.org). T678_R679delinsKK has been demonstrated to confer Tazemetostat (EPZ-6438) resistance in culture (PMID: 28231254), but has not been biochemically characterized and therefore, its effect on Ezh2 protein function is unknown (PubMed, Apr 2020).||Y|
|Molecular Profile||Indication/Tumor Type||Response Type||Therapy Name||Approval Status||Evidence Type||Efficacy Evidence||References|
|EZH2 T678_R679delinsKK||acute myeloid leukemia||resistant||Tazemetostat||Preclinical - Cell culture||Actionable||In a preclinical study, a murine acute myeloid leukemia cell line expressing EZH2 T678_R679delinsKK were resistant to Tazemetostat (EPZ-6438) in culture (PMID: 28231254).||28231254|
|DOT1L V293_L296delinsMM||acute myeloid leukemia||decreased response||Pinometostat||Preclinical - Cell culture||Actionable||In a preclinical study, a murine acute myeloid leukemia cell line expressing DOT1L V293_L296delinsMM demonstrated reduced sensitivity to EPZ-5676 (pinometostat) in culture (PMID: 28231254).||28231254|